While many studies of the past years have focused on the derivation, propagation and in vitro differentiation of human embryonic stem cells (hESC), little is known about the self-renewal and pluripotency of hESC in the aged, as opposed to the young, systemic and local organ environments. If the therapeutic hope for these cells and their progeny is to contribute to the ailing tissues in older individuals, but their regenerative capacity is adversely affected by the aged milieu, then their therapeutic value becomes significantly diminished. Unless, of course, the age-related changes affecting stem cell regenerative capacity are understood and countered. Our data strongly suggest that the aged niche has indeed a pronounced inhibitory influence on the regenerative capacity of hESC and that extrinsic cues regulating activation of stem cells become altered with age. It is, therefore, quite possible that the age-related changes in the systemic and organ environments would also preclude a productive repair of old ailing tissues by the transplanted hESC or their progeny. This work will test the specific hypothesis that the ability of hESC to regenerate skeletal muscle is, to a large extent, dependent on the age of their extrinsic environment, will define the changes in hESC regenerative and myogenic potential that are caused by the age-related alterations of their extrinsic niche, will compare the gene expression profile of hESC exposed to "young" versus "old" milieu and will provide initial molecular characterization of the inhibitory components affecting regenerative potential of stem cells in aged tissues. We will use the Federally approved hESC lines WA07 and UC06 for this proposed work. The data will help to understand the molecular mechanism(s) by which local and systemic environments control behavior of stem cells in young versus old organisms. The outcome of these studies is expected to be fundamentally important for deciphering key molecular determinants of aging, for understanding the pathways regulating hESC proliferation and cell-fate determination, and for enhancing the therapeutic value of hESC in the context of aged organs and tissues. [unreadable] [unreadable] [unreadable]